Modulation of excitability in Aplysia tail sensory neurons by tyrosine kinases

Tyrosine kinases have recently been shown to modulate synaptic plasticity and ion channel function. We show here that tyrosine kinases can also modulate both the baseline excitability state of Aplysia tail sensory neurons (SNs) as well as the excitability induced by the neuromodulator serotonin (5HT). First, we examined the effects of increasing and decreasing tyrosine kinase activity in the SNs. We found that tyrosine kinase inhibitors decrease baseline SN excitability in addition to attenuating the increase in excitability induced by 5HT. Conversely, functionally increasing cellular tyrosine kinase activity in the SNs by either inhibiting opposing tyrosine phosphatase activity or by direct injection of an active tyrosine kinase (Src) induces increases in SN excitability in the absence of 5HT. Second, we examined the interaction between protein kinase A (PKA), which is known to mediate 5HT-induced excitability changes in the SNs, and tyrosine kinases, in the enhancement of SN excitability. We found that the tyrosine kinases function downstream of PKA activation since tyrosine kinase inhibitors reduce excitability induced by activators of PKA. Finally, we examined the role of tyrosine kinases in other forms of 5HT-induced plasticity in the SNs. We found that while tyrosine kinase inhibitors attenuate excitability produced by 5HT, they have no effect on short-term facilitation (STF) of the SN-motor neuron (MN) synapse induced by 5HT. Thus tyrosine kinases modulate different forms of SN plasticity independently. Such differential modulation would have important consequences for activity-dependent plasticity in a variety of neural circuits.     

The Critical Role of Intracellular Calcium in the Mechanisms of Plasticity of Common Snail Defensive Behavior Command Neurons LPl1 and RPl1 in Nociceptive Sensitization

Studies on semi-intact common snail preparations addressed the involvement of intracellular calcium in changes in the excitability and responses to sensory stimuli of defensive behavior command neurons LPl1 and RPl1 during the acquisition of nociceptive sensitization. Application of sensitizing stimuli to the heads of control snails led to depolarization of neuron membranes, increases in neuron excitability, and depression of the responses of neurons to sensory stimuli during the short-term stage, and marked facilitation of responses in the long-term stage of sensitization. Acquisition of sensitization during profound hyperpolarization of neurons led to suppression of the increase in excitability, along with depression of responses to chemical stimulation of the head in the short- and long-term stages of sensitization. Neuron responses to tactile stimulation of the head and foot showed synaptic facilitation, similar to that seen in neurons of control animals. Acquisition of sensitization during intracellular injection of the calcium chelators EGTA and BAPTA led to suppression of synaptic facilitation in the responses of neurons to both chemical and tactile stimulation. In these conditions, membrane excitability increased to a greater extent than in neurons of control animals. The results of these experiments suggest that changes in responses to sensory stimulation in sensitized snails are associated with postsynaptic calcium-dependent mechanisms of plasticity in neurons LPl1 and RPl1.     

Selective Effects of Antibodies to Protein SMP-69 on the Activity of Defensive Behavior Command Neurons in the Common Snail

The effects of antibodies to the serotonin-modulated protein SMP-69 on the activity of defensive behavior command neurons LP11 and RP11 in semi-intact preparations from common snails were studied. Antibody to SMP-69 increased membrane excitability and facilitated neuron responses to chemical sensory stimulation by application of dilute quinine solution to the animal's head, these effects being seen at 1-1.5 h. The synaptic effects of the antibodies were specific, as they had no influence on responses induced by tactile stimulation of the head. The neuronal effects of antibody SMP-69 were similar to changes in the activity of cells LP11 and RP11 induced by serotonin and cAMP, and to changes seen when snails acquired nociceptive sensitization. It seems likely that a protein homologous to mammalian SMP-69 is involved in the mechanisms controlling excitability and long-term specific plasticity of the synaptic inputs to neurons LP11 and RP11 from chemoreceptors on the snail's head.     

Kinase-dependent modification of dendritic excitability after long-term potentiation

Patterns of presynaptic activity properly timed with postsynaptic action potential output can not only increase the strength of synaptic inputs but can also increase the excitability of dendritic branches of adult CA1 pyramidal neurons. Here, we examined the role of protein kinase A (PKA) and mitogen-activated protein kinase (MAPK) in the enhancement of dendritic excitability that occurs during theta-burst pairing of presynaptic and postsynaptic firing activity. Using dendritic and somatic whole-cell recordings in rat hippocampal slices, we measured the increase in the amplitude of back-propagating action potentials in the apical dendrite that occurs in parallel with long-term potentiation (LTP) of synaptic inputs. We found that inhibition of the MAPK pathway prevents this enhancement of dendritic excitability using either a weak or strong LTP induction protocol, while synaptic LTP can still be induced by the strong protocol. Both forms of plasticity are blocked by inhibition of PKA and occluded by interfering with cAMP degradation, consistent with a PKA-mediated increase in MAPK activity following induction of LTP. This provides a signalling mechanism for plasticity of dendritic excitability that occurs during neuronal activity and demonstrates the necessity of MAPK activation. Furthermore, this study uncovers an additional contribution of kinase activation to plasticity that may occur during learning.     

Limited contributions of serotonin to long-term hyperexcitability of Aplysia sensory neurons

Serotonin (5-HT) has provided a useful tool to study plasticity of nociceptive sensory neurons in Aplysia. Because noxious stimulation causes release of 5-HT and long-term hyperexcitability (LTH) of sensory neuron somata and because 5-HT treatment can induce long-term synaptic facilitation of sensory neuron synapses, a plausible hypothesis is that 5-HT also induces LTH of the sensory neuron soma. Prolonged or repeated exposure of excised ganglia to 5-HT produced immediate hyperexcitability of sensory neurons that showed little desensitization, but the hyperexcitability decayed within minutes of washing out the 5-HT. Prolonged or repeated treatment of either excised ganglia or dissociated sensory neurons with various concentrations of 5-HT failed to induce significant LTH even when long-term synaptic facilitation was induced in the same preparations. Use of a high-divalent cation solution to reduce interneuron activity during 5-HT treatment failed to enable the induction of LTH in excised ganglia. Pairing 5-HT application with nerve shock failed to enhance LTH produced by nerve shock or to reveal covert LTH produced by 5-HT. The induction of LTH by nerve stimulation was enhanced rather than inhibited by treatment with methiothepin, a 5-HT antagonist reported to block various 5-HT receptors and 5-HT-induced adenylyl cyclase activation. This suggests that endogenous 5-HT may have inhibitory effects on the induction of LTH by noxious stimulation. Methiothepin blocked immediate hyperexcitability produced by exogenous 5-HT and also inhibited the expression of LTH induced by nerve stimulation when applied during testing 1 day afterward. At higher concentrations, methiothepin reduced basal excitability of sensory neurons by mechanisms that may be independent of its antagonism of 5-HT receptors. Several observations suggest that early release of 5-HT and consequent cAMP synthesis in sensory neurons is not important for the induction of LTH by noxious stimulation, whereas later release of 5-HT from persistently activated modulatory neurons, with consequent elevation of cAMP synthesis, may contribute to the maintenance of LTH.     

Serotonin persistently activates the extracellular signal-related kinase in sensory neurons of Aplysia independently of cAMP or protein kinase C

Activation of the extracellular signal-related kinase is important for long-term increases in synaptic strength in the Aplysia nervous system. However, there is little known about the mechanism for the activation of the kinase in this system. We examined the activation of Aplysia extracellular signal-related kinase using a phosphopeptide antibody specific to the sites required for activation of the kinase. We found that phorbol esters led to a prolonged activation of extracellular signal-related kinase in sensory cells of the Aplysia nervous system. Surprisingly, inhibitors of protein kinase C did not block this activation. Serotonin, the physiological transmitter involved in long-term synaptic facilitation, also led to prolonged activation of extracellular signal-related kinase, but inhibitors of protein kinase A or protein kinase C did not block this activation. We examined whether the protein synthesis-dependent increase in excitability stimulated by phorbol esters was dependent on phorbol ester activation of extracellular signal-related kinase, but increases in excitability were still seen in the presence of inhibitors of extracellular signal-related kinase activation. Our results suggest that prolonged phosphorylation of extracellular signal-related kinase in the Aplysia system is not mediated by either of the classic second messenger activated kinases in this system, protein kinase A or protein kinase C and that extracellular signal-related kinase is not important for phorbol ester induced long-term effects on excitability.     

PKC modulation of transmitter release by SNAP-25 at sensory-to-motor synapses in aplysia

Activation of phosphokinase C (PKC) can increase transmitter release at sensory-motor neuron synapses in Aplysia, but the target of PKC phosphorylation has not been determined. One putative target of PKC at synapses is the synaptosomal-associated protein of 25 kDa (SNAP-25), a member of the SNARE protein complex implicated in synaptic vesicle docking and fusion. To determine whether PKC regulated transmitter release through phosphorylation of SNAP-25, we cloned Aplysia SNAP-25 and expressed enhanced green fluorescent protein (EGFP)-coupled SNAP-25 constructs mutated at the PKC phosphorylation site Ser198 in Aplysia sensory neurons. We found several distinct effects of expression of EGFP-SNAP-25 constructs. First, the rates of synaptic depression were slowed when cells contained SNAP-25 with phosphomimetic residues Glu or Asp. Second, PDBu-mediated increases in transmitter release at na?ve synapses were blocked in cells expressing nonphosphorylated-state SNAP-25. Finally, expression of EGFP-coupled SNAP-25 but not uncoupled SNAP-25 inhibited 5-HT-mediated reversal of depression and the ability of EGFP-coupled SNAP-25 to inhibit the reversal of depression was affected by changes at Ser198. These results suggest SNAP-25 and phosphorylation of SNAP-25 by PKC can regulate transmitter release at Aplysia sensory-motor neuron synapses by a number of distinct processes.     

Evolution of Learning in Three Aplysiid Species: Differences in Heterosynaptic Plasticity Contrast with Conservation in Serotonergic Pathways

We investigated the neurobiological basis of variation in sensitization between three aplysiid species: Aplysia californica , Phyllaplysia taylori and Dolabrifera dolabrifera . We tested two different forms of sensitization induced by a noxious tail shock: local sensitization, expressed near the site of shock, and general sensitization, tested at remote sites. Aplysia showed both local and general sensitization, whereas Phyllaplysia demonstrated only local sensitization, and Dolabrifera lacked both forms of learning. We then investigated a neurobiological correlate of sensitization, heterosynaptic modulation of sensory neuron excitability by tail-nerve stimulation. We found (1) an increase in sensory neuron (SN) excitability after both ipsilateral and contralateral nerve stimulation in Aplysia , (2) a smaller and shorter-lasting increase in Phyllaplysia , and (3) no effect in Dolabrifera . Because sensitization in Aplysia is strongly correlated with serotonergic (5-HT) neuromodulation, we hypothesized that the observed interspecific variation in sensitization and SN neuromodulation might be correlated with variation in the anatomy and/or functional response of the serotonergic system. However, using immunohistochemistry, we found that all three species showed a similar pattern of 5-HT innervation. Furthermore, they also showed comparable 5-HT release evoked by tail-nerve shock, as measured with chronoamperometry. These observations indicate that interspecific variation in learning is correlated with differences in SN heterosynaptic plasticity within a backgound of evolutionary conservation in the 5-HT neuromodulatory pathway. We thus hypothesize that evolutionary changes in learning phenotype do not involve modifications of the 5-HT pathway per se , but rather, changes in the response of SNs to the activation of this or other neuromodulatory pathways upon noxious stimulation.     

Endogenous PGE2 regulates membrane excitability and synaptic transmission in hippocampal CA1 pyramidal neurons

The significance of cyclooxygenases (COXs), the rate-limiting enzymes that convert arachidonic acid (AA) to prostaglandins (PGs) in the brain, is unclear, although they have been implicated in inflammatory responses and in some neurological disorders such as epilepsy and Alzheimer's disease. Recent evidence that COX-2, which is expressed in postsynaptic dendritic spines, regulates PGE2 signaling in activity-dependent long-term synaptic plasticity at hippocampal perforant path-dentate granule cell synapses, suggests an important role of the COX-2-generated PGE2 in synaptic signaling. However, little is known of how endogenous PGE2 regulates neuronal signaling. Here we showed that endogenous PGE2 selectively regulates fundamental membrane and synaptic properties in the hippocampus. Somatic and dendritic membrane excitability was significantly reduced when endogenous PGE2 was eliminated with a selective COX-2 inhibitor in hippocampal CA1 pyramidal neurons in slices. Exogenous application of PGE2 produced significant increases in frequency of firing, excitatory postsynaptic potentials (EPSP) amplitude, and temporal summation in slices treated with the COX-2 inhibitor. The PGE2-induced increase in membrane excitability seemed to result from its inhibition of the potassium currents, which in turn, boosted dendritic Ca2+ influx during dendritic-depolarizing current injections. In addition, the PGE2-induced enhancement of EPSPs was blocked by eliminating both PKA and PKC activities. These findings indicate that endogenous PGE2 dynamically regulates membrane excitability, synaptic transmission, and plasticity and that the PGE2-induced synaptic modulation is mediated via cAMP-PKA and PKC pathways in rat hippocampal CA1 pyramidal neurons.     

In vitro conditioning induces morphological changes in Hermissenda type B photoreceptor

Short- and long-term synaptic plasticity are considered to be cellular substrates of learning and memory. The mechanisms underlying synaptic plasticity especially with respect to morphology, however, are not known. In vitro conditioning in molluscan preparations is a well established form of short-term synaptic plasticity. Five paired presentations of light and vestibular stimulation to the isolated nervous system of Hermissenda results in an increase in excitability of the identified neuron, the type B photoreceptor, indicated by 2 measures, an increase in the input resistance and a cumulative depolarization after the cessation of light stimulus recorded from the cell soma. The terminal branches of type B photoreceptors iontophoretically injected with fluorescent dye were analyzed using computer-aided 3-dimensional reconstruction of images obtained using a confocal microscope under 'blind' conditions. The terminal branches contracted along the centro-lateral axis within an hour after conditioning, paralleling the increase in neuronal excitability. These data suggest that in vitro conditioning in Hermissenda is a form of short-term synaptic plasticity that involves changes in macromolecular synthesis.     

去除感觉神经元胞体促进离体培养的海兔感觉神经突触形成

目的 研究去除离体培养的海兔感觉神经元（SN）与运动神经元（MN,L7）互相接触形成的突触终末数量的变化。方法 体外培养无脊椎海生动物海兔（Aplysia）SN和MN,L7,两者互相接触形成突触联系fSN／L7）,培养到第4d时,将部分感觉神经细胞的体部去除（SN／L7,-CB）,其中一部分应用5-HT处理,另一部分应用5-HT＋茴香霉素（Anisomysin）处理,将仍保留细胞体部（SN／L7,＋CB）的离体培养突触作为对照组,24h后。利用免疫组织化学方法观察突触终末数目的变化。结果与对照组相比,应用5-HT后24h,5-HT处理组突触终末的数目显著增加（P〈0．01）;而5-HT＋茴香霉素组的突触终末数目却没有明显改变。结论 在长时程易化条件下,去除感觉神经细胞体可能使突触终末的数量增加,而这种感觉神经突触可塑性的变化可以被蛋白质合成抑制剂茴香霉素所抵消。     

c-Jun N-terminal phosphorylation is essential for hippocampal synaptic plasticity

c-Jun N-terminal kinase (JNK), a member of the MAPK family, is an important regulatory factor of synaptic plasticity as well as neuronal differentiation and cell death. Recently, JNK has been reported to modulate synaptic plasticity by the direct phosphorylation of synaptic proteins. The specific role of c-Jun phosphorylation in JNK mediated synaptic plasticity, however, remains unclear. In this study, we investigated the effects of c-Jun phosphorylation on synaptic structure and function by using c-Jun mutant mice, c-JunAA, in which the active phosphorylation sites at serines 63 and 73 were replaced by alanines. The gross hippocampal anatomy and number of spines on hippocampal pyramidal neurons were normal in c-JunAA mice. Basal synaptic transmission, input–output ratios, and paired-pulse facilitation (PPF) were also no different in c-JunAA compared with wild-type mice. Notably, however, the induction of long-term potentiation (LTP) at hippocampal CA3–CA1 synapses in c-JunAA mice was impaired, whereas induction of long-term depression (LTD) was normal. These data suggest that phosphorylation of the c-Jun N-terminus is required for LTP formation in the hippocampus, and may help to better characterize JNK-mediated modulation of synaptic plasticity.     

The Selective Effect of a Protein Kinase C Inhibitor on Synaptic Plasticity in Defensive Behavior Command Neurons During Development of Sensitization in the Snail

Studies of defensive behavior command neurons LP11 and RP11 in semi-intact common snail preparations addressed the effects of the protein kinase C antagonist polymyxin B on the effect of nociceptive sensitization. Neurons in control snails responded to application of nociceptive stimuli to the head with membrane depolarization, increases in excitability, and depression of neuron responses to sensory stimulation during the short-term stage, with marked facilitation of responses during the long-term stage of sensitization. Acquisition of sensitization in the presence of polymyxin B resulted in partial suppression of responses to nociceptive stimuli. Changes in command neuron membrane excitability in these conditions, as well as changes in responses to tactile stimulation of the foot and chemical stimulation of the head, were similar to those seen in neurons of sensitized control animals. The inhibitor also had no effect on short-term depression of neuron responses induced by tactile stimulation of the head. In addition, acquisition of sensitization during administration of polymyxin B led to complete suppression of the facilitation of responses to tactile stimulation of the snail's head during the long-term stage of sensitization. It is suggested that in sensitized common snails, protein kinase C is involved in controlling the mechanisms of nociception and is also involved in the mechanisms of selective induction of plasticity in the synaptic inputs of command neurons, which are activated by tactile stimulation of the animal's head.     

Oroxylin A increases BDNF production by activation of MAPK-CREB pathway in rat primary cortical neuronal culture

Oroxylin A (5,7-dihydroxy-6-methoxyfavone) is a flavonoid compound originated from the root of Scutellaria baicalensis Georgi. Our previous reports suggested that oroxylin A improves memory function in rat, at least in part, by its antagonistic effects on GABA(A) receptor. In addition, oroxylin A protects neurons from ischemic damage by mechanisms currently not clear. In this study we determined whether oroxylin A modulates the level of brain derived neurotrophic factor (BDNF) in primary rat cortical neuronal culture, which is well known for its role on neuronal survival, neurogenesis, differentiation of neurons and synapses and learning and memory. Treatment of oroxylin A for 3-48h increased BDNF expression which was analyzed by ELISA assay and Western blot analysis. Oroxylin A induced slow but sustained increases in intracellular calcium level and activated ERK1/2 mitogen activated protein kinase (MAPK). In addition, oroxylin A phosphorylated cyclic AMP response element binding protein (CREB) at Ser 133 in concentration and time dependent manner. Pretreatment with the MAPK inhibitor PD98059 (10μM) attenuated phosphorylation of ERK1/2 and CREB as well as BDNF production, which suggests that oroxylin A regulates BDNF production by activating MAPK-CREB pathway. GABA(A) antagonist bicuculline mimicked the effects of oroxylin A on BDNF production as well as MAPK-CREB pathway. Increase in intracellular Ca(2+) concentration, phosphorylation of ERK1/2 and CREB, and BDNF expression by oroxylin A was blocked by NMDA receptor inhibitor MK-801 (10μM) as well as tetrodotoxin (TTX, 0.5 and 1μM). The results from the present study suggest that the calcium and p-CREB dependent induction of BDNF expression, possibly via activation of synaptic NMDA receptor through the blockade of GABA(A) activity in cortical neuronal circuitry, might be responsible for the neuroprotective or memory enhancing effects of oroxylin A.     

Activin induces long-lasting N-methyl-D-aspartate receptor activation via scaffolding PDZ protein activin receptor interacting protein 1

Calcium entry into the postsynaptic neuron through N-methyl-D-aspartate-type glutamate receptors (NMDARs) triggers the induction of long-term potentiation (LTP), which is considered to contribute to synaptic plasticity and plays a critical role in behavioral learning. We report here that activin, a member of the transforming growth factor-beta (TGF-beta) superfamily, promotes phosphorylation of NMDARs and increases the Ca2+ influx through these receptors in primary cultured rat hippocampal neurons. This signal transduction occurs in a functional complex of activin receptors, NMDARs, and Src family tyrosine kinases, including Fyn, formed on a multimer of postsynaptic scaffolding postsynaptic density protein 95/Dlg/ZO-1 (PDZ), activin receptor interacting protein 1 (ARIP1). Activin-induced NMDAR activation persists for more than 24 h, which is complimentary to the activation time of NMDARs by brain-derived neurotrophic factor (BDNF). Our results suggest that activin is a unique and powerful potentiator for NMDAR-dependent signaling, which could be involved in the regulatory mechanisms of synaptic plasticity.     

Activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway leading to cyclic AMP response element-binding protein phosphorylation is required for the long-term facilitation process of aversive olfactory learning in young rats

The mitogen-activated protein kinase/extracellular-signal regulated kinase (MAPK/ERK) cascade is an important contributor to synaptic plasticity that underlies learning and memory. ERK activation by the MAPK/ERK kinase (MEK) leading to cyclic-AMP response element binding protein (CREB) phosphorylation is implicated in the formation of long-term memory. We have demonstrated that CREB phosphorylation in the olfactory bulb (OB) is important for aversive olfactory learning in young rats, yet whether MAPK/ERK functions as an upstream regulator are necessary for this olfactory learning remains to be determined. Therefore, we addressed this issue using behavioral and Western blot analyses. The MEK inhibitor PD98059 was continuously infused into the OB of postnatal day 11 rat pups during a 30-min training session regarding the pairing of citral odor and foot shock. On the following day, the time spent in the part of the apparatus where the odor was present was measured as an index of odor aversion. PD98059 impaired olfactory learning in a dose-dependent manner without affecting memory retention 1 h after training. We further tested whether odor-shock training leads to MAPK/ERK activation in the OB and defines the time course of the activation. Phosphorylated ERKs (P-ERKs) 1 and 2 were significantly increased for 60 min after the training without changes in total ERKs 1 and 2. By contrast, intrabulbar infusion of PD98059 during the training significantly reduced P-ERKs 1 and 2 as well as phosphorylated CREB without any effects on the total ERKs or CREB. Taken together with the previous findings, these results indicate that the MAPK/ERK-CREB pathway is required for the long-term, but not the short-term, facilitation process of aversive olfactory learning in young rats.     

Spike timing-dependent plasticity: a learning rule for dendritic integration in rat CA1 pyramidal neurons

Long-term plasticity of dendritic integration is induced in parallel with long-term potentiation (LTP) or depression (LTD) based on presynaptic activity patterns. It is, however, not clear whether synaptic plasticity induced by temporal pairing of pre- and postsynaptic activity is also associated with synergistic modification in dendritic integration. We show here that the spike timing-dependent plasticity (STDP) rule accounts for long-term changes in dendritic integration in CA1 pyramidal neurons in vitro . Positively correlated pre- and postsynaptic activity (delay: +5/+50 ms) induced LTP and facilitated dendritic integration. Negatively correlated activity (delay: −5/−50 ms) induced LTD and depressed dendritic integration. These changes were not observed following positive or negative pairing with long delays (> ±50 ms) or when NMDA receptors were blocked. The amplitude–slope relation of the EPSP was facilitated after LTP and depressed after LTD. These effects could be mimicked by voltage-gated channel blockers, suggesting that the induced changes in EPSP waveform involve the regulation of voltage-gated channel activity. Importantly, amplitude–slope changes induced by STDP were found to be input specific, indicating that the underlying changes in excitability are restricted to a limited portion of the dendrites. We conclude that STDP is a common learning rule for long-term plasticity of both synaptic transmission and dendritic integration, thus constituting a form of functional redundancy that insures significant changes in the neuronal output when synaptic plasticity is induced.